Arachidonic acid is converted into a variety of physiologically potent, oxygenated metabolites including prostaglandins, thromboxanes, prostacyclin, and leukotrienes. The two enzymes which most closely control these conversions, cyclooxygenase and lipoxygenase, catalyze the incorporation of molecular oxygen into the fatty acid such that the primary products contain the peroxide bond. These primary products are key intermediates in that they occur at branch points in the metabolic sequence and are further converted into compounds of widely differing, even opposed, physiological activities. As a result, the biosynthesis and further metabolism of these peroxide containing compounds play a mediating role in medically important processes i.e., hemostasis and immunity. Despite the pivotal role of these compounds, information regarding their concentration levels in physiological fluids is not presently available. The reason for this is that these determinations present such a challenging analytical problem. The peroxides lack the stability needed for many laboratory manipulations and may be present in exceedingly small quantities. The objective of the research contained in this proposal is to quantify the peroxide intermediates of arachidonate metabolism in cells of the circulatory system. The combination of two recently developed analytical tools, high performance liquid chromatography and electrochemical detection, will provide the solution to this problem. The peroxides will be separated by the unique resolving power of high performance liquid chromatography, while electrochemical detection will provide both the sensitivty and selectivity for identifying the components. This development will make it possible to evaluate the metabolic partitioning of arachidonic acid between the cyclooxygenase and 1 ipoxygenase pathways directly. Such information will be of diagnostic value for diseases which affect the arachidonate cascade. Further, a knowledge of the levels of these compounds will make it possible to monitor the effects of therapeutic agents on arachidonic acid metabolism.